The endothelial cells of the blood vessel intima produce a relaxing factor (or factors), which is not a prostanoid, both under basal conditions and during stimulation with substances such as acetylcholine. Despite many investigations, the nature, of the relaxing factor(s); the mechanism of its production/release and its mode of action on the vascular smooth muscle cells have not been determined. Most of the uncertainties regarding the cellular mechanism(s) underlying endothelium-dependent vascular responses stems from the fact that it is difficult to affect the production and the action of the factor(s) separately. Based on earlier attempts, a bioassay technique has been developed which allows separate analysis of the nature, the production and the action on smooth muscle of endothelium-derived relaxing factor(s). Rings of canine coronary, femoral and pulmonary arteries and saphenous and pulmonary veins and of the thoracic aorta of rats and rabbits with or without endothelium will be suspended for isometric tension recording in organ chambers. In the bioassay apparatus, blood vessel segments, with and without endothelium, will be perfused with modified Krebs-Ringer solution; the perfusate will be bioassayed using blood vessel rings without endothelium. Separate treatment of the perfused segments or bioassay rings with various scavengers or activators of oxygen-derived free radicals will determine whether or not these radicals play a role in the production or the action on smooth muscle of endothelium-derived relaxing factor(s). Analyzing in a similar manner (a) the ability of acetylcholine to release relaxing factor(s) from different blood vessels; (b) the ability of various substances to release relaxing factor(s) from the same blood vessel; and (c) the ability of the same relaxing factor to inhibit contractions in various bioassay rings, will determine whether the vascular endothelium releases one or more relaxing factor(s). The effects of inhibition or stimulation of Na+,K+-ATPase in the endothelium and smooth muscle will reveal how alterations in Na+,K+-exchanges modify the production or the action of endothelium-derived relaxing factor(s). The proposed research will improve the understanding of the role of endothelial cells in controlling vascular tone. It should also provide new information on pharmacological modulation of endothelium-dependent phenomena and indicate the potential role of the endothelium in the pathophysiology of vascular diseases.